Formulation of human antibodies for treating TNF-alpha associated disorders

ABSTRACT

A liquid aqueous pharmaceutical formulation is described which has a high protein concentration, a pH of between about 4 and about 8, and enhanced stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/095,393, filed Apr. 11, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/826,357, filed Aug. 14, 2015, now U.S. Pat. No.9,327,032, issued May 3, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/558,182, filed Dec. 2, 2014, now U.S. Pat. No.9,114,166, issued Aug. 25, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/453,490, filed Aug. 6, 2014, now U.S. Pat. No.8,916,158, issued Dec. 23, 2014, which is a continuation of U.S. patentapplication Ser. No. 14/322,581, filed Jul. 2, 2014, now U.S. Pat. No.8,911,741, issued Dec. 16, 2014, which is continuation of U.S. patentapplication Ser. No. 14/091,938, filed Nov. 27, 2013, now U.S. Pat. No.8,795,670, issued Aug. 5, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/471,820, filed May 15, 2012, now U.S. Pat. No.8,932,591, issued Jan. 13, 2015, which is a continuation of U.S. patentapplication Ser. No. 10/525,292 filed Oct. 27, 2005, now U.S. Pat. No.8,216,583, issued Jul. 10, 2012, which is a United States National StageApplication under 35 U.S.C. §371 of PCT/IB2003/004502, filed Aug. 15,2003 (now expired), which is a continuation of U.S. patent applicationSer. No. 10/222,140, filed Aug. 16, 2002 (now abandoned). Each of theseapplications is herein incorporated by reference in its entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 110222-0005-321-SL.txt. The text file is 11,490bytes in size, was created on Jan. 27, 2017, and is being submittedelectronically via EFS Web.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α (TNFα) is a cytokine produced by numerous celltypes, including monocytes and macrophages, that was originallyidentified based on its capacity to induce the necrosis of certain mousetumors (see e.g., Old, L. (1985) Science 230:630-632). Subsequently, afactor termed cachectin, associated with cachexia, was shown to be thesame molecule as TNFα. TNFα has been implicated in mediating shock (seee.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518;Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655).Furthermore, TNFα has been implicated in the pathophysiology of avariety of other human diseases and disorders, including sepsis,infections, autoimmune diseases, transplant rejection andgraft-versus-host disease (see e.g., Moeller, A., et al. (1990) Cytokine2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European PatentPublication No. 260 610 B1 by Moeller, A., et al. Vasilli, P. (1992)Annu. Rev. Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994)Annu. Rev. Med. 45:491-503).

Because of the harmful role of human TNFα (hTNFα) in a variety of humandisorders, therapeutic strategies have been designed to inhibit orcounteract hTNFα activity. In particular, antibodies that bind to, andneutralize, hTNFα have been sought as a means to inhibit hTNFα activity.Some of the earliest of such antibodies were mouse monoclonal antibodies(mAbs), secreted by hybridomas prepared from lymphocytes of miceimmunized with hTNFα (see e.g., Hahn T; et al., (1985) Proc Natl AcadSci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys. Res.Commun. 137:847-854; Hirai, M., et al. (1987) J. Immunol. Methods96:57-62; Fendly, B. M., et al. (1987) Hybridoma 6:359-370; Moeller, A.,et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller etal.; European Patent Publication No. 186 833 B1 by Wallach, D.; EuropeanPatent Application Publication No. 218 868 A1 by Old et al.; EuropeanPatent Publication No. 260 610 B1 by Moeller, A., et al.). While thesemouse anti-hTNFα antibodies often displayed high affinity for hTNFα(e.g., Kd≦10⁻⁹M) and were able to neutralize hTNFα activity, their usein vivo may be limited by problems associated with administration ofmouse antibodies to humans, such as short serum half life, an inabilityto trigger certain human effector functions and elicitation of anunwanted immune response against the mouse antibody in a human (the“human anti-mouse antibody” (HAMA) reaction).

In an attempt to overcome the problems associated with use offully-murine antibodies in humans, murine anti-hTNFα antibodies havebeen genetically engineered to be more “human-like.” For example,chimeric antibodies, in which the variable regions of the antibodychains are murine-derived and the constant regions of the antibodychains are human-derived, have been prepared (Knight, D. M, et al.(1993) Mol. Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 byDaddona, P. E., et al.). Additionally, humanized antibodies, in whichthe hypervariable domains of the antibody variable regions aremurine-derived but the remainder of the variable regions and theantibody constant regions are human-derived, have also been prepared(PCT Publication No. WO 92/11383 by Adair, J. R., et al.). However,because these chimeric and humanized antibodies still retain some murinesequences, they still may elicit an unwanted immune reaction, the humananti-chimeric antibody (HACA) reaction, especially when administered forprolonged periods, e.g., for chronic indications, such as rheumatoidarthritis (see e.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127;Elliot, M. J., et al. (1994) Lancet 344:1105-1110).

A preferred hTNFα inhibitory agent to murine mAbs or derivatives thereof(e.g., chimeric or humanized antibodies) would be an entirely humananti-hTNFα antibody, since such an agent should not elicit the HAMAreaction, even if used for prolonged periods. Human monoclonalautoantibodies against hTNFα have been prepared using human hybridomatechniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568; Boyle,P., et al. (1993) Cell. Immunol. 152:569-581; European PatentApplication Publication No. 614 984 A2 by Boyle, et al.). However, thesehybridoma-derived monoclonal autoantibodies were reported to have anaffinity for hTNFα that was too low to calculate by conventionalmethods, were unable to bind soluble hTNFα and were unable to neutralizehTNFα-induced cytotoxicity (see Boyle, et al.; supra). Moreover, thesuccess of the human hybridoma technique depends upon the naturalpresence in human peripheral blood of lymphocytes producingautoantibodies specific for hTNFα. Certain studies have detected serumautoantibodies against hTNFα in human subjects (Fomsgaard, A., et al.(1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog.Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H-G., etal. (1991) J. Immunol. Methods 139:145-147).

Alternative to naturally-occurring human anti-hTNFα antibodies would bea recombinant hTNFα antibody. Recombinant human antibodies that bindhTNFα with relatively low affinity (i.e., K_(d)˜10⁻⁷M) and a fast offrate (i.e., K_(off)˜10⁻² sec⁻¹) have been described (Griffiths, A. D.,et al. (1993) EMBO J. 12:725-734). However, because of their relativelyfast dissociation kinetics, these antibodies may not be suitable fortherapeutic use. Additionally, a recombinant human anti-hTNFα has beendescribed that does not neutralize hTNFα activity, but rather enhancesbinding of hTNFα to the surface of cells and enhances internalization ofhTNFα (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45; PCTPublication No. WO 92/03145 by Aston, R. et al.)

Recombinant human antibodies that bind soluble hTNFα with high affinityand slow dissociation kinetics and that have the capacity to neutralizehTNFα activity, including hTNFα-induced cytotoxicity (in vitro and invivo) and hTNFα-induced cell activation, have also been described (seeU.S. Pat. No. 6,090,382).

SUMMARY OF THE INVENTION

There is a need for a stable aqueous pharmaceutical formulation with anextended shelf life, comprising an antibody which is suitable fortherapeutic use to inhibit or counteract detrimental hTNFα activity.There is also a need for a stable aqueous pharmaceutical formulationwith an extended shelf life, comprising an antibody suitable fortherapeutic use which is easily administered and contains a high proteinconcentration.

This invention provides a liquid aqueous pharmaceutical formulationconsisting of a therapeutically effective amount of an antibody in abuffered solution forming a formulation having a pH between about 4 andabout 8 and having a shelf life of at least 18 months. The inventionalso includes an aqueous pharmaceutical formulation comprising atherapeutically effective amount of an antibody in a buffered solutionforming a formulation having a pH between about 4 and 8 and having ashelf life of at least 18 months in the liquid state. In one embodiment,the pharmaceutical formulation has enhanced stability. In a furtherembodiment, the formulation of the invention is stable following atleast 3 freeze/thaw cycles of the formulation. In another embodiment,the antibody is directed to TNFα. In yet another embodiment, theantibody is directed to human TNFα. In still another embodiment, theantibody is D2E7.

This invention also provides a liquid aqueous pharmaceutical formulationcomprising a therapeutically effective amount of an antibody in abuffered solution forming a formulation having a pH between 4 and 8 andhaving enhanced stability of at least 12 months at a temperature of 2-8°C. In one embodiment, the formulation has enhanced stability of at least18 months. In another embodiment, the antibody is directed to TNFα. Inyet another embodiment, the antibody is directed to human TNFα. In afurther embodiment, the antibody is D2E7.

The invention further provides a liquid aqueous pharmaceuticalformulation comprising a therapeutically effective amount of an antibodyin a buffered solution forming a formulation having a pH between about 4and about 8 which is easily administratable. In one embodiment, theantibody is directed to TNFα. In yet another embodiment, the antibody isdirected to human TNFα. In a further embodiment, the antibody is D2E7.

In one embodiment of the invention, the liquid aqueous pharmaceuticalformulation is suitable for injection. In a further embodiment, theformulation is suitable for single use sc injection. In anotherembodiment, the concentration of the antibody in the liquid aqueouspharmaceutical formulation is about 1-150 mg/ml. In yet anotherembodiment, the concentration of the antibody in the formulation isabout 50 mg/ml. In still another embodiment, the formulation has a highprotein concentration. In yet another embodiment of the invention, theformulation is not light sensitive.

In one embodiment of the invention, the liquid aqueous pharmaceuticalformulation contains an antibody, or an antigen-binding portion thereof,that dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and neutralizes human TNFα cytotoxicity in a standardin vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less. In anotherembodiment, the formulation of the invention contains an antibody, orantigen-binding portion thereof, which dissociates from human TNFα witha K_(off) rate constant of 5×10⁻⁴ s⁻¹ or less. In a further embodiment,the formulation contains an antibody, or antigen-binding portionthereof, which dissociates from human TNFα with a K_(off) rate constantof 1×10⁻⁴ s⁻¹ or less. In still a further embodiment, the formulation ofthe invention contains an antibody, or antigen-binding portion thereof,which neutralizes human TNFα cytotoxicity in a standard in vitro L929assay with an IC₅₀ of 1×10⁻⁸ M or less. In yet another embodiment of theinvention, the claimed formulation includes an antibody, orantigen-binding portion thereof, which neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁹ Mor less. Another embodiment of the invention, includes a formulationwhere the antibody, or antigen-binding portion thereof, neutralizeshuman TNFα cytotoxicity in a standard in vitro L929 assay with an IC₅₀of 1×10⁻¹⁰ M or less.

In another embodiment of the invention, the liquid aqueouspharmaceutical formulation contains of an antibody, or antigen-bindingportion thereof, which is a recombinant antibody, or antigen-bindingportion thereof. In another embodiment, the formulation contains anantibody, or antigen-binding portion thereof, which inhibits humanTNFα-induced expression of ELAM-1 on human umbilical vein endothelialcells. In still another embodiment, the claimed formulation includes theD2E7 antibody.

In another embodiment of the invention, the liquid aqueouspharmaceutical formulation includes an antibody, or antigen-bindingportion, thereof which dissociates from human TNFα with a K_(off) rateconstant of 1×10⁻³ s⁻¹ or less, as determined by surface plasmonresonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12. In another embodiment, the formulation of theinvention includes an antibody, or an antigen-binding portion thereof,which dissociates from human TNFα with a K_(off) rate constant of 5×10⁻⁴s⁻¹ or less. In yet another embodiment of the invention, the formulationincludes an antibody, or an antigen-binding portion thereof, whichdissociates from human TNFα with a K_(off) rate constant of 1×10⁻⁴ s⁻¹or less.

In yet another embodiment of the invention, the liquid aqueouspharmaceutical formulation, contains of an antibody, or antigen-bindingportion thereof, which has a light chain variable region (LCVR) having aCDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, ormodified from SEQ ID NO: 3 by a single alanine substitution at position1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having aCDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, ormodified from SEQ ID NO: 4 by a single alanine substitution at position2, 3, 4, 5, 6, 8, 9, 10 or 11. In a further embodiment, the formulationof the invention contains an antibody, wherein the LCVR of the antibody,or an antigen-binding portion thereof, further has a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 5 and the HCVR of theantibody, or an antigen-binding portion thereof, further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 6. In yetanother embodiment, the formulation of the invention contains anantibody, wherein the LCVR of the antibody, or an antigen-bindingportion thereof, further has CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7 and the HCVR has a CDR1 domain comprising theamino acid sequence of SEQ ID NO: 8.

In yet another embodiment of the invention, the antibody orantigen-binding portion thereof, contained in the liquid aqueouspharmaceutical formulation has a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chainvariable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2. In another embodiment, the antibody, or antigen-binding portionthereof, has an IgG1 heavy chain constant region. In still anotherembodiment, the antibody, or antigen-binding portion thereof, has anIgG4 heavy chain constant region. In another embodiment, the antibody,or antigen-binding portion thereof, is a Fab fragment. In still afurther embodiment, the antibody, or antigen-binding portion thereof, isa single chain Fv fragment.

In one embodiment of the invention, the liquid aqueous pharmaceuticalformulation, contains an antibody, or antigen-binding portion thereof,which has a light chain variable region (LCVR) having a CDR3 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or with a heavy chain variableregion (HCVR) having a CDR3 domain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO: 33 and SEQ ID NO: 34. In still another embodiment, theantibody, or antigen-binding portion thereof, neutralizes the activityof human TNFα, chimpanzee TNFα and at least one additional primate TNFαselected from the group consisting of baboon TNFα, marmoset TNFα,cynomolgus TNFα and rhesus TNFα. In a further embodiment, theformulation of the invention includes an antibody, or an antigen-bindingportion thereof, which also neutralizes the activity of mouse TNFα. Theformulation of the invention also an antibody, or an antigen-bindingportion thereof, which neutralizes the activity of pig TNFα.

In a further embodiment, the invention provides a liquid aqueouspharmaceutical formulation which contains an antibody, orantigen-binding portion thereof, which binds to human TNFα andcomprises:

a light chain CDR3 domain comprising the amino acid sequence of SEQ IDNO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution atposition 1, 4, 5, 7 or 8 or by one to five conservative amino acidsubstitutions at positions 1, 3, 4, 6, 7, 8 and/or 9, and

a heavy chain CDR3 domain comprising the amino acid sequence of SEQ IDNO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution atposition 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservativeamino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or12. In one embodiment, the liquid aqueous pharmaceutical formulationincludes an antibody which bind human TNFα and comprises a light chainvariable region (LCVR) having a CDR3 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26 or a heavy chain variable region (HCVR) having a CDR3domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ IDNO: 34. In a further embodiment of the invention, the antibody, orantigen-binding portion thereof, binds human TNFα and is the antibodyD2E7 or an antigen binding portion thereof.

The invention also provides an aqueous pharmaceutical compositioncomprising a polyol, a surfactant, and a buffer system comprisingcitrate and/or phosphate with a pH of about 4 to 8, in amountssufficient to formulate an antibody for therapeutic use at aconcentration of greater than about 45 mg/ml. In one embodiment, thepolyol is mannitol and the surfactant is polysorbate 80. In anotherembodiment, the composition includes 5-20 mg/ml of mannitol and 0.1-10mg/ml of polysorbate 80. In a further embodiment, the compositionincludes the antibody D2E7.

The invention also provides a liquid aqueous pharmaceutical formulationconsisting of 1-150 mg/ml of antibody, 5-20 mg/ml of mannitol, 0.1-10mg/ml of Tween-80, and a buffer system comprising citrate and/orphosphate, with a pH of 4 to 8. In one embodiment, the antibody isdirected to hTNFα. In another embodiment, the formulation contains about40 mg of antibody. The invention further provides a liquid aqueouspharmaceutical formulation comprising about 50 mg/ml of antibody, about12 mg/ml of mannitol, about 1 mg/ml of Tween-80, and a buffer systemcomprising citrate and/or phosphate, with a pH of about 4 to about 8. Inone embodiment, the pH of the formulation is between about 4.5 to about6.0. In another embodiment, the pH is between about 4.8 to about 5.5. Inyet another embodiment, the pH of the invention is between about 5.0 toabout 5.2.

In one embodiment of the invention, the liquid aqueous pharmaceuticalformulation also includes about 1.305 mg/ml of citric acid, about 0.305mg/ml of sodium citrate, about 1.53 mg/ml of disodium phosphatedihydrate, about 0.86 mg/ml of sodium dihydrogen phosphate dihydrate,and about 6.165 mg/ml of sodium chloride. In another embodiment, theformulation of the invention includes an antibody which is directed tohTNFα. In yet another embodiment, the formulation of the inventionincludes the antibody D2E7. In yet a further embodiment, the formulationof the invention is administered to a subject suffering from a disorderin which TNFα activity is detrimental such that TNFα activity in thesubject is inhibited

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to a liquid aqueous pharmaceutical formulationwith a pH of about 4 to about 8 which contains a high proteinconcentration, including an antibody concentration ranging from about 1to about 150 mg/ml, and has enhanced stability. This invention alsopertains to a liquid aqueous pharmaceutical formulation for therapeuticuse in a subject suffering from a condition characterized by detrimentalTNFα activity. The formulation of the invention comprises the followingconstituents: an antibody which binds to human TNFα with high affinity,a low off rate and high neutralizing capacity; a buffer, which includescitric acid, sodium citrate, disodium phosphate dihydrate, and sodiumdihydrogen phosphate dihydrate; tonicity agents, which include mannitoland sodium chloride; a detergent, including polysorbate 80; and sodiumhydroxide, for pH adjustment.

Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

The term “subject” is intended to include living organisms, e.g.,prokaryotes and eukaryotes. Examples of subjects include mammals, e.g.,humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits,rats, and transgenic non-human animals. In specific embodiments of theinvention, the subject is a human.

The term “pharmaceutical formulation” refers to preparations which arein such form as to permit the biological activity of the activeingredients to be unequivocally effective, and which contain noadditional components which are significantly toxic to the subjects towhich the formulation would be administered. “Pharmaceuticallyacceptable” excipients (vehicles, additives) are those which canreasonably be administered to a subject mammal to provide an effectivedose of the active ingredient employed.

A “stable” formulation is one in which the antibody therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Various analytical techniques formeasuring protein stability are available in the art and are reviewed inPeptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., MarcelDekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. DrugDelivery Rev. 10: 29-90 (1993), for example. Stability can be measuredat a selected temperature for a selected time period. Preferably, theformulation is stable at room temperature (about 30° C.) or at 40° C.for at least 1 month and/or stable at about 2-8° C. for at least 1 yearfor at least 2 years. Furthermore, the formulation is preferably stablefollowing freezing (to, e.g., −70° C.) and thawing of the formulation,hereinafter referred to as a “freeze/thaw cycle.”

An antibody “retains its physical stability” in a pharmaceuticalformulation if it shows substantially no signs of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography.

An antibody “retains its chemical stability” in a pharmaceuticalformulation, if the chemical stability at a given time is such that theantibody is considered to still retain its biological activity asdefined below. Chemical stability can be assessed by detecting andquantifying chemically altered forms of the antibody. Chemicalalteration may involve size modification (e.g. clipping) which can beevaluated using size exclusion chromatography, SDS-PAGE and/ormatrix-assisted laser desorption ionization/time-of-flight massspectrometry (MALDI/TOF MS), for example. Other types of chemicalalteration include charge alteration (e.g. occurring as a result ofdeamidation) which can be evaluated by ion-exchange chromatography, forexample.

An antibody “retains its biological activity” in a pharmaceuticalformulation, if the antibody in a pharmaceutical formulation isbiologically active for its intended purpose. For example, biologicalactivity is retained if the biological activity of the antibody in thepharmaceutical formulation is within about 30%, about 20%, or about 10%(within the errors of the assay) of the biological activity exhibited atthe time the pharmaceutical formulation was prepared (e.g., asdetermined in an antigen binding assay).

“Isotonic” is a term recognized in the art. Isotonic can mean, forexample, that the formulation of interest has essentially the sameosmotic pressure as human blood. Isotonic formulations will generallyhave an osmotic pressure from about 250 to 350 mOsm. Isotonicity can bemeasured using a vapor pressure or ice-freezing type osmometer, forexample. A “tonicity agent” is a compound which renders the formulationisotonic.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and nonreducing sugars), sugar alcohols and sugaracids. Preferred polyols herein have a molecular weight which is lessthan about 600 kD (e.g. in the range from about 120 to about 400 kD). A“reducing sugar” is one which contains a hemiacetal group that canreduce metal ions or react covalently with lysine and other amino groupsin proteins and a “nonreducing sugar” is one which does not have theseproperties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Nonreducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof. Where it desired that the formulation is freeze-thawstable, the polyol is preferably one which does not crystallize atfreezing temperatures (e.g. −20° C.) such that it destabilizes theantibody in the formulation. The polyl may also act as a tonicity agent.In one embodiment of the invention, one ingredient of the formulation ismannitol in a concentration of 5 to 20 mg/ml. In a preferred embodimentof the invention, the concentration of mannitol is 7.5 to 15 mg/ml. In amore preferred embodiment of the invention, the concentration ofmannitol is 10-14 mg/ml.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention has a pH in the range from about 4 to about 8;preferably from about 4.5 to about 7; and most preferably has a pH inthe range from about 5.0 to about 6.5. Examples of buffers that willcontrol the pH in this range include acetate (e.g. sodium acetate),succinate (such as sodium succinate), gluconate, histidine, citrate andother organic acid buffers.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” or “effective amount” of an antibodyrefers to an amount effective in the prevention or treatment of adisorder for the treatment of which the antibody is effective. A“disorder” is any condition that would benefit from treatment with theantibody. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predisposes the subject tothe disorder in question.

A “preservative” is a compound which can be included in the formulationto essentially reduce bacterial action therein, thus facilitating theproduction of a multi-use formulation, for example. Examples ofpotential preservatives include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administration to mammals. The carriers include liquid orsolid filler, diluent, excipient, solvent or encapsulating material,involved in carrying or transporting the subject agent from one organ,or portion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.

The term “human TNFα” (abbreviated herein as hTNFα, or simply hTNF), asused herein, is intended to refer to a human cytokine that exists as a17 kD secreted form and a 26 kD membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of hTNFα is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment of theinvention, the formulation contains an antibody with CDR1, CDR2, andCDR3 sequences like those described in U.S. Pat. Nos. 6,090,382 and6,258,562, each incorporated by reference herein.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Inone embodiment of the invention, the formulation contains anantigen-binding portions described in U.S. Pat. Nos. 6,090,382 and6,258,562, each incorporated by reference herein.

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II, below), antibodies isolated from a recombinant,combinatorial human antibody library (described further in Section III,below), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNFα is substantially free of antibodies that specifically bindantigens other than hTNFα). An isolated antibody that specifically bindshTNFα may, however, have cross-reactivity to other antigens, such asTNFα molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to hTNFα results in inhibition of the biological activity ofhTNFα. This inhibition of the biological activity of hTNFα can beassessed by measuring one or more indicators of hTNFα biologicalactivity, such as hTNFα-induced cytotoxicity (either in vitro or invivo), hTNFα-induced cellular activation and hTNFα binding to hTNFαreceptors. These indicators of hTNFα biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art, and described in U.S. Pat. Nos. 6,090,382 and 6,258,562, eachincorporated by reference herein. Preferably, the ability of an antibodyto neutralize hTNFα activity is assessed by inhibition of hTNFα-inducedcytotoxicity of L929 cells. As an additional or alternative parameter ofhTNFα activity, the ability of an antibody to inhibit hTNFα-inducedexpression of ELAM-1 on HUVEC, as a measure of hTNFα-induced cellularactivation, can be assessed.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Jöhnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

II. Antibodies of the Formulation

The invention is directed to a liquid aqueous pharmaceutical formulationcomprising a therapeutically effective amount of an antibody in abuffered solution forming a formulation having a pH between about 4 andabout 8 and having an extended shelf life, preferably of at least about18 months. In another embodiment, the liquid aqueous pharmaceuticalformulation of the invention has enhanced stability. In a furtherembodiment of the invention, the formulation is not light sensitive. Inyet another embodiment of the invention, the claimed formulation remainsstable following at least 3 freeze/thaw cycles. In still anotherembodiment, the pharmaceutical formulation of the invention is suitablefor single use sc injection.

Antibodies that can be used in the formulation include polyclonal,monoclonal, recombinant antibodies, single chain antibodies, hybridantibodies, chimeric antibodies, humanized antibodies, or fragmentsthereof. Antibody-like molecules containing one or two binding sites foran antigen and a Fc-part of an immunoglobulin can also be used. Anexample of an antibody-like molecule is the active ingredient etanerceptor infliximab. Preferred antibodies used in the formulation are humanantibodies which are cloned from human cells or from gene-archivesrepresenting the human antibody-reservoir. Especially preferred amongthe human antibodies are antibodies directed against the antigen TNFα,including human TNFα(or hTNFα).

In one embodiment, the formulation of the invention includes acombination of antibodies (two or more), or a “cocktail” of antibodies.For example, the formulation can include the antibody D2E7 and one ormore additional antibodies.

In a preferred embodiment of the invention, the formulation contains anantibody, or antigen-binding portion thereof, dissociates from humanTNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constant of1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less. In another preferred embodiment, theformulation of the invention contains an antibody, or antigen-bindingportion thereof, like those described in U.S. Pat. Nos. 6,090,382 and6,258,562, each incorporated by reference herein.

In one aspect, the formulation of the invention contains D2E7 antibodiesand antibody portions, D2E7-related antibodies and antibody portions,and other human antibodies and antibody portions with equivalentproperties to D2E7, such as high affinity binding to hTNFα with lowdissociation kinetics and high neutralizing capacity. In another oneembodiment, the formulation of the invention contains an isolated humanantibody, or an antigen-binding portion thereof, that dissociates fromhuman TNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constantof 1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, dissociates from humanTNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, witha K_(off) of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less, even more preferably with an IC₅₀ of 1×10⁻⁹ M or less and stillmore preferably with an IC₅₀ of 5×10⁻¹⁰ M or less. In a preferredembodiment, the formulation contains an antibody which is an isolatedhuman recombinant antibody, or an antigen-binding portion thereof. Inanother preferred embodiment, the formulation contains an antibody whichalso neutralizes TNFα-induced cellular activation, as assessed using astandard in vitro assay for TNFα-induced ELAM-1 expression on humanumbilical vein endothelial cells (HUVEC).

III. Preparation of Formulation

The present invention features formulations (e.g., protein formulationsand/or antibody formulations) having improved properties as compared toart-recognized formulations. For example, the formulations of theinvention have an improved shelf life and/or stability as compared toart recognized formulations. In a preferred aspect, the formulations ofthe invention comprise a high protein concentration, including, forexample, a protein concentration greater than about 45 mg/ml, a proteinconcentration greater than about 50 mg/ml, a protein concentrationgreater than about 100 mg/ml, or a protein concentration greater thanabout 150 mg/ml. In a preferred embodiment of the invention, the proteinis an antibody. In another preferred embodiment, the antibody is D2E7.The invention also provides an aqueous pharmaceutical compositioncomprising a polyol, a surfactant, and a buffer system comprisingcitrate and/or phosphate with a pH of about 4 to 8, in amountssufficient to formulate an antibody for therapeutic use at aconcentration of greater than about, for example, 45 mg/ml.

Preparation of the antibody of interest is performed according tostandard methods known in the art. In a preferred embodiment of theinvention, the antibody used in the formulation is expressed in CHOcells and purified by a standard series of chromatography steps. In afurther preferred embodiment, the antibody is directed to hTNFα, and isprepared according to the methods described in U.S. Pat. Nos. 6,090,382and 6,258,562, each incorporated by reference herein.

After preparation of the antibody of interest, the pharmaceuticalformulation comprising the antibody is prepared. The therapeuticallyeffective amount of antibody present in the formulation is determined,for example, by taking into account the desired dose volumes and mode(s)of administration. In one embodiment of the invention, the concentrationof the antibody in the formulation is between about 1 to about 150 mg ofantibody per ml of liquid formulation. In a preferred embodiment, theconcentration of the antibody in the formulation is between about 5 toabout 80 mg per ml. In another preferred embodiment, the concentrationof the antibody in the formulation is between about 25 to about 50mg/ml. The formulation is especially suitable for large antibody dosagesof more than 15 mg/ml. In a preferred embodiment, the concentration ofthe antibody is 50 mg/ml.

In another embodiment of the invention, the concentration of theantibody in the formulation is about 1-150 mg/ml, about 5-145 mg/ml,about 10-140 mg/ml, about 15-135 mg/ml, about 20-130 mg/ml, about 25-125mg/ml, about 30-120 mg/ml, about 35-115 mg/ml, about 40-110 mg/ml, about45-105 mg/ml, about 50-100 mg/ml, about 55-95 mg/ml, about 60-90 mg/ml,about 65-85 mg/ml, about 70-80 mg/ml, or about 75 mg/ml. Rangesintermediate to the above recited concentrations, e.g., about 6-144mg/ml, are also intended to be part of this invention. For example,ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included.

In one embodiment, the invention provides a formulation with an extendedshelf life comprising of an active ingredient, preferably an antibody,in combination with mannitol, citric acid monohydrate, sodium citrate,disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate,sodium chloride, polysorbate 80, water, and sodium hydroxide. In afurther embodiment, the formulation of the invention has an extendedshelf life of at least about 18 months in the liquid state. Freezing theformulation of the invention can also be used to further extend itsshelf life.

An aqueous formulation is prepared comprising the antibody in apH-buffered solution. The buffer of this invention has a pH ranging fromabout 4 to about 8, preferably from about 4.5 to about 6.0, morepreferably from about 4.8 to about 5.5, and most preferably has a pH ofabout 5.0 to about 5.2. Ranges intermediate to the above recited pH'sare also intended to be part of this invention. For example, ranges ofvalues using a combination of any of the above recited values as upperand/or lower limits are intended to be included. Examples of buffersthat will control the pH within this range include acetate (e.g. sodiumacetate), succinate (such as sodium succinate), gluconate, histidine,citrate and other organic acid buffers.

In a preferred embodiment of the invention, the formulation comprises abuffer system which contains citrate and phosphate to maintain the pH ina range of about 4 to about 8. In a further preferred embodiment the pHrange is from about 4.5 to about 6.0, more preferably from about pH 4.8to about 5.5, and most preferably in a pH range of about 5.0 to about5.2. In another preferred embodiment, the buffer system includes citricacid monohydrate, sodium citrate, disodium phosphate dihydrate, and/orsodium dihydrogen phosphate dihydrate. In a further preferredembodiment, the buffer system includes about 1.3 mg/ml of citric acid(e.g., 1.305 mg/ml), about 0.3 mg/ml of sodium citrate (e.g., 0.305mg/ml), about 1.5 mg/ml of disodium phosphate dihydrate (e.g. 1.53mg/ml), about 0.9 mg/ml of sodium dihydrogen phosphate dihydrate (e.g.,0.86), and about 6.2 mg/ml of sodium chloride (e.g., 6.165 mg/ml). Inadditional preferred embodiments, the buffer system includes 1-1.5 mg/mlof citric acid, 0.25 to 0.5 mg/ml of sodium citrate, 1.25 to 1.75 mg/mlof disodium phosphate dihydrate, 0.7 to 1.1 mg/ml of sodium dihydrogenphosphate dihydrate, and 6.0 to 6.4 mg/ml of sodium chloride. In afurther embodiment, the pH of the formulation is adjusted with sodiumhydroxide.

A polyol, which acts as a tonicifier and may stabilize the antibody, isalso included in the formulation. The polyol is added to the formulationin an amount which may vary with respect to the desired isotonicity ofthe formulation. Preferably the aqueous formulation is isotonic. Theamount of polyol added may also alter with respect to the molecularweight of the polyol. For example, a lower amount of a monosaccharide(e.g. mannitol) may be added, compared to a disaccharide (such astrehalose). In a preferred embodiment of the invention, the polyol whichis used in the formulation as a tonicity agent is mannitol. In apreferred embodiment of the invention, the mannitol concentration isabout 5 to 20 mg/ml. In another preferred embodiment of the invention,the concentration of mannitol is about 7.5 to 15 mg/ml. In a morepreferred embodiment of the formulation of the invention, theconcentration of mannitol is about 10-14 mg/ml. In the most preferredembodiment, the concentration of mannitol is about 12 mg/ml. In anotherembodiment of the invention, the polyol sorbitol is included in theformulation.

A detergent or surfactant is also added to the antibody formulation.Exemplary detergents include nonionic detergents such as polysorbates(e.g. polysorbates 20, 80 etc) or poloxamers (e.g. poloxamer 188). Theamount of detergent added is such that it reduces aggregation of theformulated antibody and/or minimizes the formation of particulates inthe formulation and/or reduces adsorption. In a preferred embodiment ofthe invention, the formulation includes a surfactant which is apolysorbate. In another preferred embodiment of the invention, theformulation contains the detergent polysorbate 80 or Tween 80. Tween 80is a term used to describe polyoxyethylene (20) sorbitanmonooleate (seeFiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4thedi., 1996). In one preferred embodiment, the formulation containsbetween about 0.1 and about 10 mg/ml of polysorbate 80, more preferablybetween about 0.5 and about 5 mg/ml. In another preferred embodiment,about 0.1% polysorbate 80 is found in the formulation of the invention.

In a preferred embodiment of the invention, the formulation is a 0.8 mLsolution in a vial containing the ingredients shown below in Table 1.

TABLE 1 1 vial with 0.8 mL solution for injection¹⁾ contains: Name ofingredient Quantity Function Active substance: Antibody (D2E7)²⁾ 40.0 mgActive substance Excipients: Mannitol 9.6 mg Tonicity agent Citric acidmonohydrate 1.044 mg Buffer Citric acid Sodium citrate 0.244 mg BufferSodium citrate Disodium phosphate 1.224 mg Buffer dihydrate Dibasicsodium phosphate dihydrate Sodium dihydrogen 0.688 mg Buffer phosphatedihydrate Monobasic sodium phosphate dihydrate Sodium chloride 4.932 mgTonicity agent Polysorbate 80 0.8 mg Detergent Water for injections759.028-759.048 mg Solvent Water for injection Sodium hydroxide³⁾0.02-0.04 mg pH adjustment Total 817.6 mg ¹⁾Density of the solution:1.022 g/mL ²⁾Is used as concentrate ³⁾Addition as 1M solution

In one embodiment, the formulation contains the above-identified agents(i.e. antibody, buffer, polyol and detergent) and is essentially free ofone or more preservatives, such as benzyl alcohol, phenol, m-cresol,chlorobutanol and benzethonium Cl. In another embodiment, a preservativemay be included in the formulation, particularly where the formulationis a multidose formulation. One or more other pharmaceuticallyacceptable carriers, excipients or stabilizers such as those describedin Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)may be included in the formulation provided that they do notsignificantly adversely affect the desired characteristics of theformulation. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed and include;additional buffering agents; co-solvents; antioxidants includingascorbic acid and methionine; chelating agents such as EDTA; metalcomplexes (e.g. Zn-protein complexes); biodegradable polymers such aspolyesters; and/or salt-forming counterions such as sodium.

The formulation herein may also be combined with one or more othertherapeutic agents as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect the antibody of the formulation. Such therapeuticagents are suitably present in combination in amounts that are effectivefor the purpose intended. Additional therapeutic agents which can becombined with the formulation of the invention are further described inU.S. Pat. Nos. 6,090,382 and 6,258,562, each of which is incorporatedherein by reference.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, preparation of the formulation.

IV. Administration of Formulation

The formulation of the invention can be used in similar indications asthose described in U.S. Pat. Nos. 6,090,382 and 6,258,562, eachincorporated by reference herein, and further detailed below.

The language “effective amount” of the formulation is that amountnecessary or sufficient to inhibit TNFα activity, e.g., prevent thevarious morphological and somatic symptoms of a detrimental TNFαactivity-associated state. In another embodiment, the effective amountof the formulation is the amount necessary to achieve the desiredresult. In one example, an effective amount of the formulation is theamount sufficient to inhibit detrimental TNFα activity. In anotherexample, an effective amount of the formulation is 0.8 mL of theformulation containing 40 mg of antibody, as described in table 1. Theeffective amount can vary depending on such factors as the size andweight of the subject, or the type of illness. For example, the choiceof a TNFα activity-inhibiting formulation can affect what constitutes an“effective amount”. One of ordinary skill in the art would be able tostudy the aforementioned factors and make the determination regardingthe effective amount of the TNFα activity inhibiting formulation withoutundue experimentation.

The regimen of administration can affect what constitutes an effectiveamount. The TNFα activity-inhibiting formulation can be administered tothe subject either prior to or after the onset of detrimental TNFαactivity. Further, several divided dosages, as well as staggereddosages, can be administered daily or sequentially, or the dose can becontinuously infused, or can be a bolus injection. Further, the dosagesof the TNFα activity-inhibiting formulation can be proportionallyincreased or decreased as indicated by the exigencies of the therapeuticor prophylactic situation.

The term “treated,” “treating” or “treatment” includes the diminishmentor alleviation of at least one symptom associated or caused by thestate, disorder or disease being treated. For example, treatment can bediminishment of one or several symptoms of a disorder or completeeradication of a disorder.

Actual dosage levels of the active ingredients (antibody) in thepharmaceutical formulation of this invention may be varied so as toobtain an amount of the active ingredient that is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the antibody found in the formulation, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular compound employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition of the present invention required. For example, thephysician or veterinarian could start doses of the compounds of theinvention employed in the pharmaceutical formulation at levels lowerthan that required in order to achieve the desired therapeutic effectand gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a formulation of the invention willbe that amount of the formulation that is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. An effective amount of theformulation of the present invention is an amount that inhibits TNFαactivity in a subject suffering from a disorder in which TNFα activityis detrimental. In a preferred embodiment, the formulation provides aneffective dose of 40 mg per injection of the active ingredient, theantibody. In another embodiment, the formulation provides an effectivedose which ranges from about 1 to 150 mg of antibody. If desired, theeffective daily dose of the pharmaceutical formulation may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

In one embodiment of the invention, the dosage of the antibody in theformulation is between about 5 to about 80 mg. In another embodiment,the dosage of the antibody in the formulation is between about 25 toabout 50 mg. The formulation is especially suitable for large antibodydosages of more than 15 mg. In a preferred embodiment of the invention,the formulation provides an antibody at a dose of about 40 mg. Inanother preferred embodiment, the antibody is directed to TNFα. In themost preferred embodiment, the antibody is D2E7.

In one embodiment of the invention, the dosage of the antibody in theformulation is between about 1-150 mg, about 5-145 mg, about 10-140 mg,about 15-135 mg, about 20-130 mg, about 25-125 mg, about 30-120 mg,about 35-115 mg, about 40-110 mg, about 45-105 mg, about 50-100 mg,about 55-95 mg, about 60-90 mg, about 65-85 mg, about 70-80 mg, or about75 mg. In a preferred embodiment, the dosage of the antibody is 40 mg.In a further preferred embodiment, the antibody is directed to TNFα. Inthe most preferred embodiment, the antibody is D2E7. Ranges intermediateto the above recited dosages, e.g., about 2-149 mg, are also intended tobe part of this invention. For example, ranges of values using acombination of any of the above recited values as upper and/or lowerlimits are intended to be included.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

The invention provides a pharmaceutical formulation with an extendedshelf life, which, in one embodiment, is used to inhibit TNFα activityin a subject suffering from a disorder in which TNFα activity isdetrimental, comprising administering to the subject an antibody orantibody portion of the invention such that TNFα activity in the subjectis inhibited. Preferably, the TNFα is human TNFα and the subject is ahuman subject. Alternatively, the subject can be a mammal expressing aTNFα with which an antibody of the invention cross-reacts. Still furtherthe subject can be a mammal into which has been introduced hTNFα (e.g.,by administration of hTNFα or by expression of an hTNFα transgene). Aformulation of the invention can be administered to a human subject fortherapeutic purposes (discussed further below). In one embodiment of theinvention, the liquid pharmaceutical formulation is easilyadministratable, which includes, for example, a formulation which isself-administered by the patient. In a preferred embodiment, theformulation of the invention is administered through sc injection,preferably single use. Moreover, a formulation of the invention can beadministered to a non-human mammal expressing a TNFα with which theantibody cross-reacts (e.g., a primate, pig or mouse) for veterinarypurposes or as an animal model of human disease. Regarding the latter,such animal models may be useful for evaluating the therapeutic efficacyof antibodies of the invention (e.g., testing of dosages and timecourses of administration).

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-TNFα antibody as describedabove.

There are numerous examples of disorders in which TNFα activity isdetrimental. Examples of disorders in which TNFα activity is detrimentalare described in U.S. Application No. 60/397,275, incorporated byreference herein. Examples in which TNFα activity is detrimental arealso described in U.S. Pat. Nos. 6,015,557, 6,177,077, 6,379,666,6,419,934, 6,419,944, 6,423,321, and 6,428,787; U.S. Patent ApplicationNos. US2001/0016195, US2001/0004456, and US2001/026801; WO 00/50079 andWO 01/49321, each incorporated by reference herein.

The use of the antibodies and antibody portions of the invention in thetreatment of specific disorders is discussed further below:

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology ofsepsis, with biological effects that include hypotension, myocardialsuppression, vascular leakage syndrome, organ necrosis, stimulation ofthe release of toxic secondary mediators and activation of the clottingcascade (see e.g., Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med.45:491-503; Russell, D and Thompson, R. C. (1993) Curr. Opin. Biotech.4:714-721). Accordingly, the formulation of the invention can be used totreat sepsis in any of its clinical settings, including septic shock,endotoxic shock, gram negative sepsis and toxic shock syndrome.

Furthermore, to treat sepsis, the formulation of the invention can becoadministered with one or more additional therapeutic agents that mayfurther alleviate sepsis, such as an interleukin-1 inhibitor (such asthose described in PCT Publication Nos. WO 92/16221 and WO 92/17583),the cytokine interleukin-6 (see e.g., PCT Publication No. WO 93/11793)or an antagonist of platelet activating factor (see e.g., EuropeanPatent Application Publication No. EP 374 510).

Additionally, in a preferred embodiment, the formulation of theinvention is administered to a human subject within a subgroup of sepsispatients having a serum or plasma concentration of IL-6 above 500 pg/ml,and more preferably 1000 pg/ml, at the time of treatment (see PCTPublication No. WO 95/20978 by Daum, L., et al.).

B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in thepathophysiology of a variety of autoimmune diseases. For example, TNFαhas been implicated in activating tissue inflammation and causing jointdestruction in rheumatoid arthritis (see e.g., Tracey and Cerami, supra;Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum. 38:151-160; Fava, R.A., et al. (1993) Clin. Exp. Immunol. 94:261-266). TNFα also has beenimplicated in promoting the death of islet cells and in mediatinginsulin resistance in diabetes (see e.g., Tracey and Cerami, supra; PCTPublication No. WO 94/08609). TNFα also has been implicated in mediatingcytotoxicity to oligodendrocytes and induction of inflammatory plaquesin multiple sclerosis (see e.g., Tracey and Cerami, supra). Chimeric andhumanized murine anti-hTNFα antibodies have undergone clinical testingfor treatment of rheumatoid arthritis (see e.g., Elliott, M. J., et al.(1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet344:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.34:334-342).

The formulation of the invention can be used to treat autoimmunediseases, in particular those associated with inflammation, includingrheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and goutyarthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmuneuveitis and nephrotic syndrome. Typically, the formulation isadministered systemically, although for certain disorders, localadministration of the antibody or antibody portion at a site ofinflammation may be beneficial (e.g., local administration in the jointsin rheumatoid arthritis or topical application to diabetic ulcers, aloneor in combination with a cyclohexane-ylidene derivative as described inPCT Publication No. WO 93/19751).

C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of infectious diseases. For example, TNFαhas been implicated in mediating brain inflammation and capillarythrombosis and infarction in malaria (see e.g., Tracey and Cerami,supra). TNFα also has been implicated in mediating brain inflammation,inducing breakdown of the blood-brain barrier, triggering septic shocksyndrome and activating venous infarction in meningitis (see e.g.,Tracey and Cerami, supra). TNFα also has been implicated in inducingcachexia, stimulating viral proliferation and mediating central nervoussystem injury in acquired immune deficiency syndrome (AIDS) (see e.g.,Tracey and Cerami, supra). Accordingly, the antibodies, and antibodyportions, of the invention, can be used in the treatment of infectiousdiseases, including bacterial meningitis (see e.g., European PatentApplication Publication No. EP 585 705), cerebral malaria, AIDS andAIDS-related complex (ARC) (see e.g., European Patent ApplicationPublication No. EP 230 574), as well as cytomegalovirus infectionsecondary to transplantation (see e.g., Fietze, E., et al. (1994)Transplantation 58:675-680). The formulation of the invention, also canbe used to alleviate symptoms associated with infectious diseases,including fever and myalgias due to infection (such as influenza) andcachexia secondary to infection (e.g., secondary to AIDS or ARC).

D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograftrejection and graft versus host disease (GVHD) and in mediating anadverse reaction that has been observed when the rat antibody OKT3,directed against the T cell receptor CD3 complex, is used to inhibitrejection of renal transplants (see e.g., Tracey and Cerami, supra;Eason, J. D., et al. (1995) Transplantation 59:300-305; Suthanthiran, M.and Strom, T. B. (1994) New Engl. J. Med. 331:365-375). Accordingly, theformulation of the invention, can be used to inhibit transplantrejection, including rejections of allografts and xenografts and toinhibit GVHD. Although the antibody or antibody portion may be usedalone, more preferably it is used in combination with one or more otheragents that inhibit the immune response against the allograft or inhibitGVHD. For example, in one embodiment, the formulation of the inventionis used in combination with OKT3 to inhibit OKT3-induced reactions. Inanother embodiment, the formulation of the invention is used incombination with one or more antibodies directed at other targetsinvolved in regulating immune responses, such as the cell surfacemolecules CD25 (interleukin-2 receptor-α), CD11a (LFA-1), CD54 (ICAM-1),CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet anotherembodiment, the formulation of the invention is used in combination withone or more general immunosuppressive agents, such as cyclosporin A orFK506.

E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia,stimulating tumor growth, enhancing metastatic potential and mediatingcytotoxicity in malignancies (see e.g., Tracey and Cerami, supra).Accordingly, the formulation of the invention, can be used in thetreatment of malignancies, to inhibit tumor growth or metastasis and/orto alleviate cachexia secondary to malignancy. The formulation may beadministered systemically or locally to the tumor site.

F. Pulmonary Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofadult respiratory distress syndrome, including stimulatingleukocyte-endothelial activation, directing cytotoxicity to pneumocytesand inducing vascular leakage syndrome (see e.g., Tracey and Cerami,supra). Accordingly, the formulation of the invention, can be used totreat various pulmonary disorders, including adult respiratory distresssyndrome (see e.g., PCT Publication No. WO 91/04054), shock lung,chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonaryfibrosis and silicosis. The formulation may be administered systemicallyor locally to the lung surface, for example as an aerosol.

G. Intestinal Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofinflammatory bowel disorders (see e.g., Tracy, K. J., et al. (1986)Science 234:470-474; Sun, X-M., et al. (1988) J. Clin. Invest.81:1328-1331; MacDonald, T. T., et al. (1990) Clin. Exp. Immunol.81:301-305). Chimeric murine anti-hTNFα antibodies have undergoneclinical testing for treatment of Crohn's disease (van Dullemen, H. M.,et al. (1995) Gastroenterology 109:129-135). The formulation of theinvention, also can be used to treat intestinal disorders, such asidiopathic inflammatory bowel disease, which includes two syndromes,Crohn's disease and ulcerative colitis.

H. Cardiac Disorders

The formulation of the invention, also can be used to treat variouscardiac disorders, including ischemia of the heart (see e.g., EuropeanPatent Application Publication No. EP 453 898) and heart insufficiency(weakness of the heart muscle)(see e.g., PCT Publication No. WO94/20139).

I. Others

The pharmaceutical formulation of the invention, also can be used totreat various other disorders in which TNFα activity is detrimental.Examples of other diseases and disorders in which TNFα activity has beenimplicated in the pathophysiology, and thus which can be treated usingthe formulation of the invention, include inflammatory bone disordersand bone resorption disease (see e.g., Bertolini, D. R., et al. (1986)Nature 319:516-518; Konig, A., et al. (1988) J. Bone Miner. Res.3:621-627; Lerner, U. H. and Ohlin, A. (1993) J. Bone Miner. Res.8:147-155; and Shankar, G. and Stern, P. H. (1993) Bone 14:871-876),hepatitis, including alcoholic hepatitis (see e.g., McClain, C. J. andCohen, D. A. (1989) Hepatology 9:349-351; Felver, M. E., et al. (1990)Alcohol. Clin. Exp. Res. 14:255-259; and Hansen, J., et al. (1994)Hepatology 20:461-474) and viral hepatitis (Sheron, N., et al. (1991) J.Hepatol. 12:241-245; and Hussain, M. J., et al. (1994) J. Clin. Pathol.47:1112-1115), coagulation disturbances (see e.g., van der Poll, T., etal. (1990) N. Engl. J. Med. 322:1622-1627; and van der Poll, T., et al.(1991) Prog. Clin. Biol. Res. 367:55-60), burns (see e.g., Giroir, B.P., et al. (1994) Am. J. Physiol. 267:H118-124; and Liu, X. S., et al.(1994) Burns 20:40-44), reperfusion injury (see e.g., Scales, W. E., etal. (1994) Am. J. Physiol. 267:G1122-1127; Serrick, C., et al. (1994)Transplantation 58:1158-1162; and Yao, Y. M., et al. (1995)Resuscitation 29:157-168), keloid formation (see e.g., McCauley, R. L.,et al. (1992) J. Clin. Immunol. 12:300-308), scar tissue formation;pyrexia; periodontal disease; obesity and radiation toxicity.

Other disorders in which TNFα activity is detrimental include, but arenot limited to, adult Still's disease, Alzheimer's disease, ankylosingspondylitis, asthma, cancer and cachexia, atherosclerosis, chronicatherosclerosis, chronic fatigue syndrome, liver failure, chronic liverfailure, obstructive pulmonary disease, chronic obstructive pulmonarydisease, congestive heart failure, dermatopolymyositis, diabeticmacrovasculopathy, endometriosis, familial periodic fevers, fibrosis,hemodialysis, Jarisch-Herxheimer reaction, juvenile RA, Kawasakisyndrome, myelo dysplastic syndrome, myocardial infarction,panciaticular vulgaris, periodontal disease, peripheral neuropathy,polyarticular, polymyositis, progressive renal failure, psoriasis,psoriatic arthritis, Reiter's syndrome, sarcoidosis, scleroderma,spondyloarthropathies, Still's disease, stroke, therapy associatedsyndrome, therapy induced inflammatory syndrome, inflammatory syndromefollowing IL-2 administration, thoracoabdominal aortic aneurysm repair(TAAA), Vasulo-Behcet's disease, Yellow Fever vaccination, type 1diabetes mellitus, type 2 diabetes mellitus, neuropathic pain, sciatica,cerebral edema, edema in and/or around the spinal cord, vasculitide,Wegener's granulomatosis, temporal arteritis, polymyalgia rheumatica,Takayasu's arteritis, polyarteritis nodosa, microscopic polyangiitis,Churg-Strauss syndrome, Felty's syndrome, Sjogren's syndrome, mixedconnective tissue disorder, relapsing polychondritis, pseudogout,loosening of prostheses, autoimmune hepatitis, sclerosing cholangitis,acute pancreatitis, chronic pancreatitis, glomerulonephritides,post-streptococcal glomerulonephritis or IgA nephropathy, rheumaticheart disease, cardiomyopathy, orchitis, pyoderma gangerenosum, multiplemyeloma, TNF receptor associated periodic syndrome [TRAPS],atherosclerosis, steroid dependent giant cell arteritismyostitis,uveitis, and drug reactions.

The invention is further illustrated in the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1: Preparation of the Formulation

The pharmaceutical formulation of the invention was made according tothe following protocol.

Materials which were used in the formulation include: mannitol, citricacid monohydrate (citric acid), sodium citrate, disodium phosphatedihydrate (dibasic sodium phosphate dihydrate), sodium dihydrogenphosphate dihydrate (monobasic sodium phosphate dihydrate), sodiumchloride, polysorbate 80, water for the injections, sodium hydroxide,which was used as a 1M solution to adjust the pH, and proteinconcentrate (e.g., antibody concentrate).

Preparation of 20 L of Buffer (Equivalent to 20.180 kg—Density of theSolution: 1.009 g/ml)

Ingredients were weighed out as follows: 240.0 g mannitol, 26.1 g citricacid monohydrate, 6.1 g sodium citrate, 30.6 g disodium phosphatedihydrate, 17.2 g sodium dihydrogen phosphate dihydrate, 123.3 g sodiumchloride, 20.0 g polysorbate 80, and 19,715.7 to 19,716.1 g of water.

A sodium hydroxide solution was prepared by combining 40.0 g of sodiumhydroxide with 1000.8 g of water for injections.

Next, a buffer was prepared by dissolving the following pre-weighedingredients (described above) in about 90% of the water for injections:mannitol, citric acid monohydrate, sodium citrate, disodium phosphatedihydrate, sodium dihydrogen phosphate, sodium chloride, and polysorbate80. It was determined that the sequence of the addition of the bufferconstituents was not important and can, therefore, be chosen at will.

Following addition of all of the buffer constituents, the pH of thesolution was adjusted with 1M sodium hydroxide which was prepared asdescribed above. After the addition of the sodium hydroxide, the finalweight of the water was added. The buffer solution was then filteredthrough a sterilized filter (hydrophilic polyvinylidene difluoride, 0.22μm pore size) into a sterilized receptacle. The filtration medium usedwas filtration sterilized nitrogen.

Preparation of 40 L of Formulation (Equivalent to 40.88 kg)

The filtered buffer solution was then added to the thawed and pooledantibody concentrate (the active ingredient of the pharmaceuticalformulation), prepared as follows. The antibody (concentrate) was thawedin a water bath prior to the preparation of the pharmaceuticalformulation. 34.207 g of antibody concentrate was used, which isequivalent to 2.0 kg of protein with 60 mg protein/mL proteinconcentrate. The density of the concentrate was 1.0262 g/mL. Any proteinconcentrate ranging from 25.655 to 37.316, which is equivalent to aprotein concentration in the protein concentrate of 55 to 80 mg/mL, canbe used. The buffer was added while stirring, until the final weight ofthe bulk solution was reached.

The formulation, with all of its ingredients included, was thensterilized by filtration as described above, except the formulation wasfiltered through two sterile 0.22 μm membrane filters. Followingsterilization, the formulation was packaged for use in either a vial ora pre-filled syringe.

The skilled artisan will also appreciate that the weight quantitiesand/or weight-to-volume ratios recited herein, can be converted to molesand/or molarities using the art-recognized molecular weights of therecited ingredients. Weight quantities exemplified herein (e.g., g orkg) are for the volumes (e.g., of buffer or pharmaceutical formulation)recited. The skilled artisan will appreciate that the weight quantitiescan be proportionally adjusted when different formulation volumes aredesired. For example, 32 L, 20 L, 10 L, 5 L, or 1 L formulations wouldinclude 80%, 50%, 25%, 12.5%, or 2.5%, respectively, of the exemplifiedweight quantities.

Example 2: Freeze/Thaw Studies

After the formulation buffer for the D2E7 antibody was selected the drugsubstance was formulated in the same matrix as the finished product.

Freeze thaw behavior of the D2E7 antibody drug substance at a proteinconcentration of 63 mg/mL was evaluated by cycling drug substance 3times from the frozen state to the liquid state. Table N shows theresults of an experiment evaluating the effect of three fast and slowfreeze-thaw cycles in the presence and absence of 0.1% polysorbate 80starting from −80° C. or −30° C., respectively.

Table 2 shows that the D2E7 antibody drug substance can be thawed/frozenat least 3 times without any detrimental effect on either chemical(cation exchange HPLC, size exclusion HPLC, colour, pH), physicochemicalproperties (subvisible particles, clarity) or biological activity (invitro TNF neutralization assay). Also table 2 shows that the inclusionof polysorbate 80 improved the physicochemical properties of the D2E7antibody drug substance as evidenced by the lower number of subvisibleparticles regardless whether a slow or fast freeze/thaw cycle was beingused (see table 2).

TABLE 2 Effect of freeze thaw on the D2E7 antibody drug substancewith/without polysorbate 80 Fast Fast thaw thaw No Slow thaw −30° C. inSlow thaw −80° C. in Polysorbate freeze/ −30° C. in water −80° in waterTest criteria (0.1%)¹⁾ thaw refrigerator bath refrigerator bath Clarity− 25.0 22.5 25.3 25.8 25.6 + 27.8 28.1 28.2 28.0 28.1 Colour − ≦B9 ≦B9≦B9 ≦B9 ≦B9 + ≦B9 ≦B9 ≦B9 ≦B9 ≦B9 pH − 5.01 5.02 5.02 5.02 5.02 + 5.025.02 5.02 5.02 5.02 Subvisible − 42 600 303 1891 303 particles 2 4 5 80 + 0 5 1 0 8 0 0 0 0 1 Size exclusion − 99.8 99.8 99.8 99.8 99.8 HPLC +99.8 99.8 99.8 99.8 99.8 Cation − 87.1 87.0 87.2 86.9 86.9 exchange +86.8 87.0 87.1 87.3 86.8 HPLC In vitro TNF − 118.0 123.8 118.0 103.3120.5 neutralization + 111.8 96.2 100.9 96.7 95.8 test ¹⁾+ = formulationwith 0.1% polysorbate 80; − = formulation without 0.1% polysorbate 80

Example 3: Microbial Studies

Tests were performed to determine if the formulation can supportmicrobial growth. The results from these experiments showed that theformulation does not support microbial growth if stored at 20 to 25° C.for 14 days. This result was determined by directly inoculating thesterile formulation with microorganisms (e.g., Staphylococous aureus,ATCC-No.: 6538P, Candida albicans, ATCC-No.: 10231, Aspergillus niger,ATCCC-No.: 16404, Pseudomonas aeruginosa, ATCC-No.: 9027, anenvironmental isolate) at low level (NMT 100 cfu/mL). Inoculatedformulations were then examined for overall microbial growth, e.g., forchanges in turbidity. A lack of turbidity was an indication of nooverall growth, and was detected in the inoculated containers after 14days. Further, no organisms could be reisolated from these containers.Thus it was concluded that the formulation does not support microbialgrowth under these conditions.

INCORPORATION BY REFERENCE

The contents of all references and patents cited herein are herebyincorporated by reference in their entirety.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A stable liquid aqueous pharmaceuticalformulation comprising (a) a human IgG1 anti-human Tumor Necrosis Factoralpha (TNFα) antibody at a concentration of 45-105 mg/ml, (b) a polyol,(c) a polysorbate, and (d) a buffer system comprising histidine andhaving a pH of 4 to 8, wherein the antibody is D2E7, and wherein theformulation is suitable for subcutaneous injection.
 2. The formulationof claim 1, wherein the pH is from 4.5 to 6.0.
 3. The formulation ofclaim 1, wherein the pH is from 5.0 to about 6.5.
 4. The formulation ofclaim 1, wherein the polysorbate is polysorbate 20 or polysorbate
 80. 5.The formulation of claim 4, wherein the polysorbate is polysorbate 20.6. The formulation of claim 4, wherein the polysorbate is polysorbate80.
 7. The formulation of claim 6, wherein the polyol is a sugar.
 8. Theformulation of claim 7, wherein the polyol is trehalose.
 9. Theformulation of claim 8, wherein the polysorbate 80 concentration isbetween 0.1 and 10 mg/ml.
 10. The formulation of claim 8, wherein the pHis from 4.5 to 6.0, and the antibody is at a concentration of 50 mg/ml.11. The formulation of claim 10, wherein the pH is 5.2.
 12. Theformulation of claim 10, wherein the polysorbate 80 concentration isbetween 0.1 and 10 mg/ml.
 13. The formulation of claim 7, wherein thepolyol is sucrose.
 14. The formulation of claim 13, wherein thepolysorbate 80 concentration is between 0.1 and 10 mg/ml.
 15. Theformulation of claim 13, wherein the pH is from 4.5 to 6.0, and theantibody is at a concentration of 50 mg/ml.
 16. The formulation of claim15, wherein the pH is 5.2.
 17. The formulation of claim 15, wherein thepolysorbate 80 concentration is between 0.1 and 10 mg/ml.
 18. Theformulation of claim 6, wherein the polyol is a sugar alcohol.
 19. Theformulation of claim 18, wherein the polyol is mannitol.
 20. Theformulation of claim 19, wherein the polysorbate 80 concentration isbetween 0.1 and 10 mg/ml.
 21. The formulation of claim 19, wherein thepH is from 4.5 to 6.0, and the antibody is at a concentration of 50mg/ml.
 22. The formulation of claim 21, wherein the pH is 5.2.
 23. Theformulation of claim 21, wherein the polysorbate 80 concentration isbetween 0.1 and 10 mg/ml.
 24. The formulation of claim 18, wherein thepolyol is sorbitol.
 25. The formulation of claim 24, wherein thepolysorbate 80 concentration is between 0.1 and 10 mg/ml.
 26. Theformulation of claim 24, wherein the pH is from 4.5 to 6.0, and theantibody is at a concentration of 50 mg/ml.
 27. The formulation of claim26, wherein the pH is 5.2.
 28. The formulation of claim 26, wherein thepolysorbate 80 concentration is between 0.1 and 10 mg/ml.
 29. Theformulation of claim 1, wherein the formulation is stable at 2-8° C. for1 year.
 30. The formulation of claim 1, wherein the formulation isstable following freezing to −70° C. and thawing of the formulationthree times.